(T0930-10-58) Hazard Analysis of Amorphous Silica Nanoparticle via Acquired Immune System

Purpose: The development of products containing nanoparticles has expanded significantly in various industries such as cosmetics, foods, and medicine. As the size of these particles decreases, their reactivity at the tissue interface and their tissue penetration increases. On the other hand, reduction of particle size could occur the differences in their kinetics compared to conventional materials, and their differences have been concerned to result in unexpected biological effects. Thus, we need to identify potential sources of unpredictable adverse biological effects due to increasingly smaller nanoparticles. From this viewpoint, biological evaluation of nanoparticles focusing on innate immunity and inflammatory response has been reported. However, many studies have focused on innate immunity, and information on whether nanoparticles induce biological responses through effects on acquired immunity, which is activated after innate immunity and mediates immunological memory, is sparse. Amorphous silica nanoparticles are nanoparticles that people are frequently and repeatedly exposed to because they are components of daily products. By focusing on their potential to induce various acute toxicities, we have sought to define the effects of multiple exposure of amorphous silica nanoparticles on the acquired immune system. Here, we used amorphous silica nanoparticles as model nanoparticles to assess their effects on acute hepatotoxicity as mediated through acquired immunity and the mechanism through which these effects were achieved.

Methods: BALB/c mice and C.B-17 SCID mice (n = 5-15) were intradermally pretreated with PBS or amorphous silica nanoparticles that were 50 nm in diameter (nSP50) once a week for a total of 4 times (pretreatment). After that, acute hepatotoxicity was induced by administering an excessive amount of nSP50 from the tail vein (challenge). ALT and AST were measured at 6 h after challenge. In the cell depletion experiment, BALB/c mice were injected intraperitoneally with anti-mouse CD8 antibody or isotype control antibody at 1 wk after pretreatment. For the IFN-γ blockade experiment, BALB/c mice were injected intraperitoneally with anti-mouse IFN-γ antibody or isotype control antibody at 1 wk after pretreatment. The Tukey's test was used for statistical analysis, and P < 0.05 was considered to be significantly different.

Results: Plasma levels of hepatic injury markers such as ALT and AST in BALB/c mice were significantly increased in the nSP50-pretreated/nSP50-challenged group compared with the PBS-pretreated/nSP50-challenged group. Staining with hematoxylin and eosin demonstrated that vacuolar degeneration and necrosis of hepatocytes occurred more frequently in liver sections from nSP50-pretreated mice than from PBS-pretreated mice, indicating that pretreatment with nSP50 biochemically and pathologically exacerbated nSP50-induced hepatic damage in immunocompetent mice. On the other hand, in immunodeficient C.B-17/SCID mice, the resulting plasma levels of ALT and AST did not differ significantly between the PBS-pretreated/nSP50-challenged and nSP50-pretreated/nSP50-challenged groups. These results suggest that nSP50-inducible hepatic damages would worsen by pretreatment of themselves through the acquired immunity. Then, to clarify the types of acquired immunity involved in the nSP50-induced exacerbation of hepatic damage, cell depletion analysis was performed. Plasma levels of ALT and AST of the pretreated mice with nSP50 and then treated with a neutralizing antibody to deplete CD8⁺ cells were reduced compared with those of the group treated with the isotype-matched control antibody, whereas the non-depleted group showed exacerbation of hepatic damage. The result indicates that a CD8⁺ cell–mediated immune response played a crucial role in the exacerbation of the hepatic damage induced by multiple exposure of nSP50. Subsequently, to confirm the ability of CD8⁺ T cells to be activated after nSP50 pretreatment, we placed splenocytes in medium with or without nSP50 and then measured the concentration of IFN-γ, a kind of cytokines which activates CD8⁺ T cells, in the culture supernatants. The concentrations of IFN-γ were increased in the splenocyte cultures derived from nSP50-pretreated/nSP50-challenged mice compared with those from PBS-pretreated mice. Moreover, plasma levels of ALT and AST of pretreated mice with nSP50 and then treated with a neutralizing antibody for blockade of IFN-γ activity were partially reduced compared with those of the group treated with the isotype-matched control antibody, whereas the non-blockade group showed exacerbation of hepatic damage.

Conclusion: We demonstrated that multiple exposure of nSP50 exacerbate the hepatic damage initiated by a single dose and that this exacerbation is mediated through acquired immunity. Mechanistically, CD8⁺ T lymphocytes and IFN-γ play critical roles in this process. Our current findings suggest that amorphous silica nanoparticles can activate acquired immunity and generate adverse biological effects.

References: Eto S., Higashisaka K., Koshida A., Sato K., Ogura M., Sakurai M., Tsujino H., Nagano K., Tsutsumi Y.: Amorphous silica nanoparticles exacerbate hepatic damage through the activation of acquired cell-mediated immunity., Nano Ex., 3(1): 015002, 2022.